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Abstract

Background

Maintenance of the botanical integrity of cereal kernels and the addition of acetic
acid (as vinegar) in the product or meal has been shown to lower the postprandial
blood glucose and insulin response and to increase satiety. However, the mechanism
behind the benefits of acetic acid on blood glucose and satiety is not clear. We hypothesized
that the gastric emptying rate could be involved. Thus, the aim of this study was
to evaluate the possible influence of maintained botanical integrity of cereals and
the presence of acetic acid (vinegar) on gastric emptying rate (GER), postprandial
blood glucose and satiety.

Methods

Fifteen healthy subjects were included in a blinded crossover trial, and thirteen
of the subjects completed the study. Equicarbohydrate amounts of the following wheat-based
meals were studied: white wheat bread, whole-kernel wheat bread or wholemeal wheat
bread served with white wine vinegar. The results were compared with a reference meal
consisting of white wheat bread without vinegar. The GER was measured with standardized
real-time ultrasonography using normal fasting blood glucose <6.1 mmol/l or plasma
glucose <7.0 mmol/l as an inclusion criterion. The GER was calculated as the percentage
change in the antral cross-sectional area 15 and 90 minutes after ingestion of the
various meals. Satiety scores were estimated and blood glucose was measured before
and 15, 30, 45, 60, 90 and 120 min after the start of the meal.

Results

The whole-kernel wheat bread with vinegar resulted in significantly higher (<0.05)
satiety than the wholemeal wheat bread and white wheat bread with vinegar and the
reference bread. Wheat fiber present in the wholemeal wheat bread, or the presence
of wheat kernels per se, did not affect the postprandial blood glucose or GER significantly
compared with white wheat bread, neither did the addition of vinegar to white bread
affect these variables. There was no correlation found between the satiety with antral
areas or GER

Conclusion

The present study shows higher satiety after a whole-kernel wheat bread meal with
vinegar. This may be explained by increased antral distension after ingestion of intact
cereal kernels but, in this study, not by a lower gastric emptying rate or higher
postprandial blood glucose response.

Trial registration

NTR1116

Background

Changing the diet can control the blood glucose level and help prevent the development
of type 2 diabetes. The American Diabetes Association recommends a reduced calorie
intake and increased intake of dietary fiber and whole grains to prevent the development
of type 2 diabetes [1]. Foods with a low glycemic index that are rich in fiber are recommended [1]. The first reported antiglycemic effect of vinegar was by Ebihara and Nakajima [2]. Vinegar decreases the glycemic index of, for example, rice in sushi by about 20–35%
[3]. Also, when white vinegar was added to cold storage potatoes as a vinaigrette sauce
the glycemic index was lowered in healthy subjects [4]. Vinegar in a salad dressing added to lettuce and ingested with white wheat bread
has been shown to reduce the blood glucose response, but the gastric emptying rate
(GER) measured by ultrasonography was not delayed [5]. When vinegar was neutralized to pH 6.0 no effects were seen on the postprandial
blood glucose response [5]. The decreased postprandial blood glucose response was explained by a mechanism related
to acidity and inhibition of digestive amylases [5]. Further, vinaigrette sauce added to a white wheat bread meal has been shown to reduce
postprandial blood glucose and insulin responses in healthy subjects; this was explained
by delayed gastric emptying, measured indirectly with paracetamol [6]. Insulin sensitivity was improved and postprandial insulin and glucose responses
were reduced in insulin-resistant subjects after a meal containing vinegar [7]. However, in healthy subjects, only the postprandial insulin response was reduced,
not the blood glucose response, after ingestion of a white bagel with apple cider
vinegar [7]. Satiety and eating behavior are complex, but play a key role in energy intake and
metabolic control in healthy subjects and in patients with diabetes. Only one previous
study on healthy subjects has been conducted to evaluate the effect of vinegar on
satiety. That study showed that white wheat bread ingested with vinegar increased
and prolonged the feeling of satiety according to a dose-response relation [8].

The term "whole grain" is often used for wholemeal products in which the structure
of the cereal grain is destroyed in the flour containing the original dietary fiber,
but also for cereal products in which a large proportion of whole cereal grains is
intact. However, there seems to be a major difference in metabolic response between
whole grain and wholemeal products. The preparation, cooking and particle size of
the grain structures may also affect the metabolic response. The glycemic index decreased
in patients with type 2 diabetes when increasing proportions of whole grain bulgur
(cracked wheat) were substituted for miller flour in bread [9]. However, in another study, the glycemic response did not differ between bulgur and
whole wheat kernels in patients with type 1 and type 2 diabetes [10]. This can be explained by the similar particle sizes of bulgur and wheat after chewing.
The particle size of wheat has been found to affect the digestion rate and metabolic
response in healthy subjects [11]. The wheat germ of the whole grain acts as a natural amylase inhibitor, which can
be destroyed during the milling of wheat into wholemeal flour [12].

Only one study has been conducted previously on the effect of whole kernels on gastric
emptying, which showed that in healthy subjects the gastric emptying, measured indirectly
with paracetamol, was not affected after meals composed of whole-kernel rye bread
or wholemeal rye bread compared to with white wheat bread [13]. A high-dietary-fiber meal was found to delay the GER, measured by ultrasonography,
in healthy subjects compared with a low-fiber meal [14]. However, another study showed that the GER following a high-fiber meal consisting
of whole wheat grain and rye bread, was not different from that following a low-fiber
meal [15]. In a previous study using the same wheat bread recipes as in this study and the
meals were ingested without vinegar, a significantly lower blood glucose response
and higher satiety after whole-kernel wheat bread than after white wheat bread was
observed [16]. However, the GER was not evaluated.

These divergent results indicate that not only glucose response, as was previously
known, but also satiety and gastric emptying rate seem to be influenced by variables
related to processing conditions and botanical structure. The effects of a combination
of vinegar and different fiber structures on the postprandial blood glucose response,
gastric emptying or satiety has, as far as we know, not been studied previously. Thus,
the aim of this study was to evaluate the possible influence of maintaining the botanical
integrity of cereals and the addition of acetic acid (as vinegar) on the GER, postprandial
blood glucose and satiety. The hypothesis was that products that delay gastric empting
rate also lead to higher satiety and decreased postprandial blood glucose.

Materials and methods

Fifteen healthy subjects were included in the study. However, one male subject was
excluded because he was found to have diabetes mellitus, and one female subject was
excluded because celiac disease was diagnosed during the study. Thirteen healthy subjects
(six men and seven women: mean age 25 ± 4 years [range 22–35 years]; mean BMI 22.8
± 3.07 kg/m2 [range 17.7–29.7 kg/m2]), without symptoms or a prior history of gastrointestinal disease, abdominal surgery
or diabetes mellitus, completed the study. The subjects were not receiving any drugs,
except two of the women who were taking birth control medication. One subject was
a smoker and none was a snuff user. None of the subjects used any drugs on the day
of the examination.

White wheat bread was made from 3700 g white wheat flour, 2000 g water and 200 g yeast.
The dough was allowed to rise for 20 min at 28°C. The dough was then divided into
440 g pieces and left to rise for a second time, for 35 min at 40°C (RH: 80%). Loaves
were baked at 210°C for 22 min with the addition of steam during the first 30 s. The
loaves were stored in a freezer at -20°C until used.

The whole-kernel wheat bread was made from 3076 g wheat kernels that were boiled for
20 min in 3076 g water and then cooled at room temperature, after which 1200 g water,
624 g white wheat flour and 200 g yeast were added. The dough was left to rise for
30 min and then divided into 580 g pieces. These pieces were then allowed to rise
for a second period of 45 min at 40°C (RH: 80%). Loaves were baked at 200°C for 45
min.

The wholemeal wheat bread was made from 3076 g milled wheat kernels, (500 g of the
flour was scaled with 1000 g boiling water) 1200 g water, 624 g white wheat flour
and 200 g yeast. The dough was left to rise for 30 min and then divided into 580 g
pieces. These were then allowed to rise for a period of 45 min at 40°C (RH: 80%).
Loaves were baked at 200°C for 45 min.

The reference and test meals contained 50 g available carbohydrates from bread products.
The content of available carbohydrates was analyzed according to Holm et al [17]. The portion size of the white wheat bread was 106.34 g and, besides 50 g available
carbohydrates, contained 2.1 g dietary fiber, 1.8 g fat and 8.3 g protein. The portion
size of the whole-kernel wheat bread was 132.66 g and contained, besides 50 g available
carbohydrates, 7.2 g dietary fiber, 2.9 g fat and 9.2 protein. The portion size of
the wholemeal wheat bread was 107.62 g, and contained 7.2 g dietary fiber, 2.9 g fat
and 9.2 g proteins, besides the 50 g available carbohydrates. We used the same baking
recipes and baking process as Liljeberg et al [16] for the white wheat reference bread and whole-kernel wheat bread; the content of
dietary fiber, fat and proteins were thus assumed to be the same as previously described
by Liljeberg et al [16]. The wholemeal wheat bread was made from the same recipe as whole-kernel wheat bread
but with milled wheat kernels. The three test meals contained one of the three kinds
of test bread dipped in 28 g white wine vinegar (5% acetic acid, pH 2.8–3 Druvan,
DR Persfood AB, Eslöv, Sweden), which is equivalent to 23 mmol acetic acid in each
test meal. Drinking water, 200 ml, was also served. The reference meal contained white
wheat bread and water, but without white wine vinegar. The test products and the reference
were served in random order during intervals of 1 week. The subjects were examined
between 8:00 and 10:00 am after an 8-h fast. Smoking was prohibited for 8 h before
and during the test. The fasting blood glucose concentration of each subject was checked
on the day of the examination to ensure that it was normal. If the subject reported
gastrointestinal symptoms (diarrhea or constipation) on the study day, the examination
was postponed. Each meal was ingested within 10 minutes.

The GER was estimated using a previously described standardized ultrasound method
[18]. The sonographic examination was performed using two different ultrasound machines
(Siemens Acuson Sequoa 512 and Aloka Prof. Sound) with a multi-MHz abdominal transducer.
The same machine was used to calculate values of GER. The measurements of the gastric
antrum were performed by the same radiologist, who was blinded with regard to the
meals. The measurements were made 15 and 90 minutes after the end of meal ingestion
(25 and 100 min after the start of the meal). Gastric emptying was expressed as the
percentage change of the antral cross-sectional area from 15 to 90 min. Paired t-test
was performed before the beginning of the study and power calculations showed a 71%
power to detect a 20% change in GER.

Finger-prick capillary samples were collected before and 15, 30, 45, 60, 90 and 120
min after the start of the meal to measure blood glucose levels. Blood glucose concentrations
were measured with a HemoCue Glucose system (HemoCue AB, Ängelholm, Sweden). The validated
satiety score scale was used according to the method of Haber et al on the basis of
a scoring system with grades from -10 (extreme hunger) to 10 (extreme satiety) [19]. Satiety scores were estimated before the meal and 15, 30, 45, 60, 90 and 120 min
after the start of the meal, using the same scoring system.

The study was performed according to the Helsinki declaration and was approved by
the Ethics Committee at Lund University, and participants provided written informed
consent.

The changes from pre-ingestion values in blood glucose and satiety after the different
treatments were presented as means ± SEMs and were tested globally in a repeated measures
linear mixed model using the interaction of time and treatment as fixed effects and
subjects as random effects (SAS, version 8.2, SAS Institute, Cary, NC). For the covariance
structure of the repeated measures within a series a spatial exponential model was
used. The areas under the curve (AUCs) above zero for delta blood glucose and satiety
responses of the four treatments were determined for each subject (Graph Pad PRISM,
version 4, San Diego, CA) and presented as means ± SEMs. These were tested globally
in a mixed model where treatments were entered as fixed effects and subjects were
entered as random effects. Tukey's multiple comparisons test was used as follow-up
procedure after the mixed models when appropriate. In addition we tested the inclusion
of BMI as covariate in the mixed model for glucose and also the possible correlation
between satiety and antral areas or GER. Median values and quartiles are presented
for the antral cross-sectional areas and the GER. These were tested globally using
the Friedman rank sum test, and when the null hypothesis was rejected, followed by
pair-wise comparisons using Wilcoxon rank sum test with the Holm sequential procedure
for P-value adjustment (R, version 2.6, The R foundation for statistical computing
c/o Institut für Statistik und Wahrscheinlichkeitstheorie, Technische Universität
Wien, Wiedner Hauptstraée 8-10/1071,1040 Vienna, Austria). Statistical significance
was accepted at p < 0.05.

Results

Postprandial blood glucose response

The mean fasting blood glucose level before the reference meal of white bread was
4.4 ± 0.2 mmol/l compared and not significant different to before ingestion of vinegar
together with white wheat bread, wholemeal bread and whole-kernel bread were 4.5 ±
0.1, 4.6 ± 0.1 and 4.5 ± 0.2 mmol min/l, respectively. No significant differences
were seen in blood glucose responses at different times, or in the incremental areas
under the postprandial glucose curves between the different bread meals (Figure 1). The mean blood glucose AUC 0–120 min after ingestion of the reference meal of white
wheat bread was 147 ± 14 mmol min/l. The AUCs after ingestion of vinegar together
with white wheat bread, wholemeal bread and whole-kernel bread were 114 ± 12, 110
± 10 and 135 ± 13 mmol min/l, respectively. The blood glucose AUCs did not differ
significantly between the meals, (p = 0.13 in the test of the global hypothesis).
The inclusion of BMI as a covariate in the analysis of postprandial blood glucose
response did not improve the model.

Figure 1.The mean (± SEM) incremental blood glucose concentration in thirteen healthy subjects
after the ingestion of meals consisting of white wheat bread only (reference) (diamond),
white wheat bread with vinegar (square), wholemeal wheat bread with vinegar (triangel)
and whole-kernel bread with vinegar (dot). No significant differences were found between the incremental blood glucose concentrations
following the various meals.

Gastric emptying rate

No significant differences were observed between the meals with regard to gastric
emptying rates (Figure 3). The median value of the GER after the reference meal was estimated to be 51% (q1
= 40%, q3 = 61%) compared with the corresponding value after the reference meal with
vinegar, which was estimated to be 47% (q1 = 36%, q3 = 56%). The median value of the
GER after the wholemeal wheat bread with vinegar meal was estimated to be 62% (q1
= 39%, q3 = 74%) which can be compared with the median value of the GER after the
whole-kernel wheat bread with vinegar meal, of 43% (q1 = 39%, q3 = 53%).

Figure 3.Gastric emptying rate following the ingestion of white wheat bread with vinegar, wholemeal
bread with vinegar, whole-kernel wheat bread with vinegar and white wheat bread (reference),
in thirteen healthy subjects. The median, minimum (Min), and maximum (Max) values and the values of the first (q1)
and the third (q3) quartiles are shown. No significant differences were found between
the GERs.

The median values of the antral cross-sectional area after the ingestion of the reference
meal were 525 mm2 (q1 = 431 mm2, q3 = 707 mm2) and 295 mm2 (q1 = 193 mm2, q3 = 364 mm2) 15 and 90 min, respectively, after the end of the meal. The median values of the
antral cross-sectional area after the ingestion of the reference meal with vinegar
were 607 mm2 (q1 = 607 mm2, q3 = 1092 mm2) and 317 mm2 (q1 = 264 mm2, q3 = 507 mm2), 15 and 90 min, respectively after the end of the meal. The median values of the
antral cross-sectional area after the ingestion of the wholemeal wheat bread with
vinegar were 660 mm2 (q1 = 531 mm2, q3 = 885 mm2) and 266 mm2 (q1 = 166 mm2, q3 = 422 mm2), respectively, 15 and 90 min after the end of the meal. After the ingestion of the
whole-kernel wheat bread with vinegar the median values of the antral cross-sectional
area were 857 mm2 (q1 = 657 mm2, q3 = 1057 mm2) and 477 mm2 (q1 = 329 mm2, q3 = 558 mm2), respectively, 15 and 90 min after the end of the meal. The median value of the
early antral cross-sectional area after the whole-kernel wheat bread with vinegar
(857 mm2) was significantly larger (p < 0.05 in a pairwise comparison using Wilcoxon rank
sum test after the global Friedman ranks sum test being significant p = 0.0022) than
the corresponding area after ingestion of the reference meal (525 mm2) (Figure 4).

Figure 4.Median antral area in thirteen healthy subjects 15 and 90 minutes after the end of
meal ingestion meals consisting of white wheat bread (reference), white wheat bread
with vinegar, wholemeal bread with vinegar, and whole-kernel wheat bread with vinegar. * Significantly different from the response to the white wheat bread (reference)
(p < 0.05).

Discussion

The aim of this study was to elucidate the effect of maintained botanical structure
and dietary fiber present in wheat-based bread products in combination with vinegar,
on gastric emptying rate, glycemic response and satiety in healthy subjects. Our hypothesis
was that an intake of intact cereal kernels with vinegar would increase satiety and
lower the postprandial blood glucose response due to delayed gastric emptying. We
were not able to verify this hypothesis. The results showed a significant increase
in satiety after ingestion of the whole-kernel wheat bread with vinegar compared with
the other meals, but no statistically significant differences were seen in gastric
emptying rate or postprandial blood glucose response. However, the antral cross-sectional
area was significantly larger 15 min after the ingestion of whole-kernel wheat bread
with vinegar than after the white wheat reference bread. Thus, the distension of the
antrum may explain the increase in satiety scores reported after the whole-kernel
wheat bread meal with vinegar compared to the other bread meals with vinegar. However,
the antral cross-sectional area did not correlate to the satiety scores. Clearly,
a larger trial involving a greater number of subjects would be needed to validate
the findings of this small study. A limitation of this study is that the sample size
was small. The effects of whole-kernal bread without vinegar were not investigated.
However, the white wheat reference bread served with vinegar and compared to white
wheat bread without vinegar did not affect the satiety. In a previous study using
the same bread recipes but without vinegar, a significantly increased satiety after
whole-kernel wheat bread than after white wheat bread was observed [16]. Therefore, it can be assumed that the whole-kernel bread was more satiating than
the other meals regardless of adding vinegar. This study thus shows that the botanical
structure rather than the amount of fiber per se causes the distension of the antrum
and increased satiety. This relationship between antral area and satiety in healthy
subjects has been observed previously by others [20-24]. Holt et al have also reported an association between the particle size of wheat
and satiety [25].

Another intention of the current study was to evaluate the effect of whole kernels
on blood glucose response and gastric emptying. This lack of difference in postprandial
blood glucose response between the bread meals was most unexpected as, in a previous
study using the same bread recipes but without vinegar, it was observed a significantly
lower blood glucose response after whole-kernel wheat bread than after white wheat
bread [16]. The botanical integrity of the grain kernels may have been unintentionally destroyed
during the baking process, which would explain the present observations. However,
the structure of the bread was not investigated. The lack of difference in GER between
the bread meals is in agreement with studied performed by Juntunen et al, who compared
whole-kernel rye bread and wholemeal rye bread to white wheat bread, despite the known
difference in insulin response between rye and wheat [13]. Unfortunately, we did not control the subjects for exercise or food choice the night
before of the testing. This may have affected the postprandial blood glucose responses.

Another intention of the current study was to evaluate the effect of vinegar on blood
glucose response and gastric emptying. However, we did not observe the lowering effect
of vinegar on blood glucose response reported with white wheat bread [8] and potatoes [4] in previous studies. However, when 20 g apple cider vinegar was ingested prior to
a low-glycemic meal the postprandial insulin response was lower, but no effect was
observed on the blood glucose response in healthy subjects [26]. However, ingestion of 20 g apple cider vinegar prior to a high-glycemic meal composed
of bagel, butter and orange juice, reduced the postprandial blood glucose and insulin
response in healthy subjects [26]. In a recent study, the postprandial glucose and insulin responses of type 2 diabetes
patients were found not to be affected, and in healthy subjects the postprandial blood
glucose levels were not affected, but the insulin levels were reduced when apple cider
vinegar was consumed prior to the meal [27]. However, it was demonstrated in insulin-resistant subjects that the postprandial
insulin and glucose responses were reduced after drinking 20 g apple cider vinegar
prior to a meal consisting of white bagel, butter and orange juice [27].

Our findings, that there was no difference in gastric emptying rate after a meal including
vinegar, agree with those of Brighenti et al, who found no difference regarding gastric
emptying, measured by ultrasonography, after a meal with 20 ml white vinegar (16 mmol
acetic acid), although the blood glucose response was reduced in healthy subjects
[5]. They explained the lower postprandial blood glucose response as being due to a mechanism
related to acidity and the inhibition of digestive amylases. Another study showed
that an addition of 20 g white vinegar (18 mmol acetic acid) to a bread meal lowered
the postprandial blood glucose and insulin responses in healthy subject, and this
was explained by delayed gastric emptying [6]. However, gastric emptying was measured indirectly using paracetamol, which is a
less reliable method.

Conclusion

The present study shows that the post-prandial ratings of satiety were higher after
whole-kernel wheat bread meal served with vinegar. This may be explained by increased
antral distension after the ingestion of intact cereal kernels.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

JH participated in the design of the study, recruited the subjects, collected the
data, performed the statistical calculations and drafted the manuscript. PH performed
the statistical calculations and participated in drafting the manuscript. SL participated
in drafting the manuscript. GD participated in the design of the study and participated
in drafting the manuscript. OB participated in the design of the study and performed
the ultrasound examinations. LOA participated in the design of the study and in drafting
the manuscript. All authors read and approved the final manuscript. All authors lack
any conflict of interest.

Acknowledgements

We would like to thank Yvonne Granfeldt and Inger Björck at the Department of Applied
Nutrition and Food Chemistry, Center for Chemistry and Chemical Engineering, University
of Lund, for assistance in the design of the study and for providing us with the bread
products and vinegar.